The overall goals of our research effort have centered on the characterization of the proteolytic degradation of native and inactivated hepatic cytochromes P450 (P450s) as models of integral endoplasmic reticulum (ER) protein degradation. These studies have revealed that all P450s (CYPs 3A, 2B1 and 2C11) inactivated via heme-modification of the protein are branded for proteolytic disposal by the cytosolic ATP- and ubiquitin (Ub)-dependent 26S proteasomal pathway, a process typical of ER-associated degradation (ERAD). In this, the CYP3A protein is phosphorylated at Ser/Thr residues by liver cytosolic kinases, ubiquitinated, dislocated from its ER-anchor and translocated to the cytosolic 26S proteasome for disposal. Our studies in the yeast Saccharomyces cerevisiae reveal that the turnover of native CYP3A4 similarly involves ERAD. While the involvement of an Ub-conjugating enzyme (Ubc7p) and the 26S proteasome in CYP3A4 ERAD has been established, the contribution of additional, recently characterized ERAD participants to CYP3A4 ubiquitination (Cue1p? Ub-ligases?) and proteasomal recognition (Cdc48p/Ufd-1p/Hrd4p?) is unknown, and a specific goal of this proposal. Similarly, the role of Thr264, Ser420 and Ser478 phosphorylation, if any, in native CYP3A4 ERAD is also unclear and another specific aim. While native CYP3A4 undergoes ERAD, native CYPs 2B1 and 2C11, its ER-counterparts, are degraded by the vacuoles/lysosomes. The reason for this differential sanitary sorting is unknown, but may lie in structural/molecular determinants "degrons" encoded in the individual P450 sequence, whose characterization is also proposed. The proposed studies rely on various analytical (HPLC-peptide mapping/mass spectrometric analyses, protein chemistry, proteomics), molecular biological (site-directed mutagenesis, chimeric plasmids), and cell biological (culture, expression, in vivo protein cross-linking) approaches. These studies center on a physiologically relevant but neglected aspect of P450 biology. Furthermore, they are focused on CYP3A4, the major human liver and intestinal enzyme, that is responsible for the metabolism of toxins, carcinogens and over 60% of clinically prescribed drugs and is particularly susceptible to this biological fate. Finally, because the 26S proteasome is responsible for the generation of antigenic peptides, these studies may provide insight into the P450 autoantibodies detected in sera of patients with chronic active and drug induced hepatitis and hypersensitivity reactions. [unreadable] [unreadable] [unreadable]